Citizens and family doctors facing awareness and management of traditional cardiovascular risk factors: results from the Global Cardiovascular Risk Reduction Project (Help Your Heart Stay Young Study).

BACKGROUND AND AIM: The numerous guidelines and multiple approaches to managing cardiovascular risk factors have reduced the number of fatal events but not the incidence of cardiovascular disease (CVD). One rarely explored aspect is the extent to which individuals perceive their own risk in relation to their education and history of CVD. Furthermore, Italy has a State-based Health System, in which family doctors (FDs) may be an extremely useful and relatively low cost resource for risk management, but the degree of their involvement in individual CVD risk management has not been previously assessed. METHODS AND RESULTS: The Department of Clinical and Experimental Medicine of Federico II University, Naples, Italy, and the Neapolitan Section of the Italian Society of Family Doctors (SIMG), developed an epidemiological survey to evaluate the level and awareness of CVD risk in subjects in the urban area of Naples, and the degree of involvement of FDs in CVD risk management. During a period of a few months, the subjects who visited their FDs were invited to respond to a standard self-explanatory questionnaire, and the FDs were required to provide quantitative information concerning the CVD risk factors of each enrolled subject from their databases in order to assess global CVD risk. The data included cholesterol and blood pressure (BP) levels, and had to be collected within six months of the visit; if the date were missing, the fact was recorded. The present analysis was based on data concerning the 5,687 subjects who had entered the study by January 2002, 7.6% of whom reported CVD (myocardial infarction (MI), stroke, angina, cerebral transient ischemic attack: CD+) and 92.4% did not (CVD-). MI was the most frequent CVD, and 18% of the CVD+ cases reported more than one non-fatal cardiovascular event. On average, the CVD+ subjects were older and more often men. After adjusting for age and FD, they also had a higher body mass index (BMI) and prevalence of obesity, higher self-reported BP, a lower education level, and more often referred high cholesterol levels, hypertension and diabetes. On the contrary, the proportion of smokers was higher in the CVD- group. Among the subjects who declared that they did not have a high cholesterol level, 11% reported recent values of > 200 mg/dL. The FDs of 36% of the cases were unable to assess the individual global CV risk level using quantitative data from their electronic databases. The most frequently missing information was the level of total cholesterol. Missing data were more frequent in the CVD- than the CVD+ subjects, regardless of age and FD. CONCLUSIONS: The results of our study suggest that the awareness of CVD risk among subjects is somewhat vague. The FDs were generally able to provide a quantitative assessment of CVD risk in their patients. CVD risk prevention programmes may be more successful if they stress knowledge and awareness in the population, and stimulate FDs to undertake more stringent quantitative assessments of CVD risk factors.

Analysis of the (CAG)n repeat at the IT15 locus in a population from Calabria (southern Italy).

The defect causing Huntington's disease (HD) has recently been discovered as an expanded CAG trinucleotide repeat located at the 5' end of the IT15 gene. This discovery allows the molecular diagnosis of HD by measuring the CAG repeat length. The normal and pathological repeat ranges in a population need to be established before a diagnostic test for HD can be performed. To determine the distribution of IT15 alleles in a population from Calabria (southern Italy), we analyzed 102 normal subjects and 9 HD patients coming from a defined area of Calabria (province of Cosenza). Expanded alleles ranged from 44 to 76 repeats. Normal alleles varied from 8 to 27 repeats, which is one of the lowest values observed at the top of the normal range; the mean was significantly different from the value observed in six other populations. The allele distribution seemed to group mainly around the mode, and no intermediate alleles were present in our sample. These results suggest a particular stability of the CAG repeat at the IT15 locus in the Calabrian group and confirm once again the peculiar genetic structure of this population.

Spectrin Cosenza: a novel beta chain variant associated with Sp alphaI/74 hereditary elliptocytosis.

A Calabrian family (Southern Italy) with Sp alpha(I/74) hereditary elliptocytosis (HE) in the heterozygous state was studied. Sp alpha(I/74) HE is associated with asymptomatic elliptocytosis, a defect in spectrin dimer self association and an increase of the alpha(I/74) kD fragment from the alpha chain after partial tryptic digestion of spectrin. To identify the underlying molecular defect, we analysed exons V, W, X, Y, Z of the beta gene and exon 2 of the alpha gene by single-strand conformational polymorphism (SSCP) of the amplification products. Direct DNA sequencing of the mutant exon showed a C-->G substitution at position 6284 of the beta gene. The corresponding substitution at the protein level was Arg-->Pro in the 2064 position of the beta-spectrin chain.

Genetic heterogeneity of glucose-6-phosphate dehydrogenase deficiency in south-east Sicily.

In order to explore the nature of glucose-6-phosphate dehydrogenase (G6PD) deficiency in south-east Sicily, we have analysed the G6PD gene in 25 unrelated males with abnormal G6PD activity and/or electrophoretic mobility, by using the analysis of the appropriate PCR-amplified fragment of DNA and subsequent digestion by appropriate restriction-enzymes, looking for the presence of certain known G6PD mutations. We amplified the entire G6PD coding sequence into eight fragments, followed by single-strand conformation polymorphism (SSCP) analysis and sequencing of those individual fragments that were found to be abnormal by SSCP. Through these methods we found a total of twelve G6PD Mediterranean variants with the association of a silent mutation 1311 (also known as polymorphic site Bcl I), one G6PD Mediterranean without this association, four G6PD A-Val 68 and two G6PD Santamaria and five G6PD Chatham. In a subject with normal activity a mutation was found in exon 5, designated as G6PD Sao Borja. This is the first report on the molecular analysis of G6PD mutations in Sicily and we have obtained evidence for four distinct classes of variants.

A simple and rapid nonisotopic method for sizing CAG repeats in the SCA1 gene.

Spinocerebellar ataxia type 1 is caused by the expansion of a CAG trinucleotide repeat, located at the 5' end of the gene responsible for the disease (SCA1 gene). We propose a simple and rapid method for SCA1 diagnosis, avoiding both radioactive and Southern blotting analysis. The method allows an accurate allele sizing by visualization of polymerase chain reaction products through a silver nitrate-stained polyacrylamide gel.

Severe erythrocyte adenylate kinase deficiency due to homozygous A-->G substitution at codon 164 of human AK1 gene associated with chronic haemolytic anaemia.

A child of Italian origin with a congenital haemolytic anaemia had spectrophotometrically undetectable erythrocyte adenylate kinase (AK) activity. Her parents and brother had approximately 50% normal AK activity, and AK electrophoresis of red blood cell (RBC) crude extract on cellulose acetate strips showed the presence of the normal allele AK1-1. No AK band was detected in the AK electrophoresis of the proband, in whom the erythrocyte 2,3-diphosphoglycerate (2,3DPG) and glutathione (GSH) concentrations were normal whereas adenosine triphosphate (ATP) concentration, pyruvate kinase (PK) and glucose-6P-dehydrogenase (G6PD) activities were increased, reflecting the high reticulocyte count (6.9%). No other evident enzymatic defect was detected by standard procedures. Analysis of AK gene exons, based on polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP), clearly showed an abnormality in the fragment containing exon 6. The subsequent sequence analysis of this abnormal fragment revealed homozygous and heterozygous A-->G substitutions in the proband and in the parents and brother respectively at codon 164, corresponding to a tyrosine-->cysteine substitution in the AK protein.

Nonisotopic method for accurate detection of (CAG)n repeats causing Huntington disease.

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded trinucleotide repeat (CAG)n located at the 5' end of the novel IT15 gene. Discovery of this expansion allows the molecular diagnosis of HD by measuring repeat length. We applied a simple nonisotopic method to detect (CAG)n repeats, avoiding both radioactive and Southern transfer analysis. The assay is based on direct visualization of electrophoresed PCR products, after silver nitrate gel staining. Its accurate sizing of HD alleles allows presymptomatic diagnosis of at-risk persons. By avoiding isotopic manipulations, the method is safe and accurate, with no radioactive background bands. Furthermore, because it permits direct allele visualization after gel staining, the method is simple and rapid, allowing allele sizing within hours rather than days.

DYS19, D12S67, and D1S80 polymorphisms in population samples from southern Italy and Greece.

Genotype and allele frequencies of the DYS19, D12S67, and D1S80 highly polymorphic loci were determined in population samples from southern Italy (103 subjects) and Greece (84 subjects) using the amplified fragment length polymorphism (AFLP) technique (polymerase chain reaction followed by native PAGE and silver staining). Five, eleven, and eighteen alleles were found at the DYS19, D12S67, and D1S80 loci, respectively. PIC values ranged from 0.55 (DYS19 locus in Italians) to 0.79 (D12S67 locus in Italians). The distribution of D12S67 and D1S80 genotypes conformed to Hardy-Weinberg equilibrium, as confirmed by three statistics. Heterogeneity G tests, carried out on allele frequency distributions, showed a significant difference between the samples at the DYS19 locus, whereas no difference was found with regard to the other polymorphisms. Using data from the literature, we widened the comparison to other European groups analyzed for the same markers. All the polymorphisms were found to distinguish between populations of the same main ethnic group. In particular, D1S80 allele frequencies distinguished the Finns from other European groups (Spanish, German, Italian, and Greek samples). The reduced assay time, the high polymorphism level, and the ability to distinguish between populations indicate that these markers have potential value in population genetic studies.

SP alpha I/65 hereditary elliptocytosis in Calabria (southern Italy).

The alpha I/65 variant of spectrin has been described in black people, in North Africans and recently in two southern Italian families. This variant is associated in the heterozygous state with mild Hereditary Elliptocytosis (HE) and the molecular basis of the defect is invariably the duplication of TTG at codon 154 of the alpha spectrin gene. The present study reports the identification of five Calabrian families with SP alpha I/65 HE and their distribution in the population.

Spectrum of beta-thalassemia mutations in Calabria: implications for prenatal diagnosis.

Using a combination of oligonucleotide probes and restriction endonuclease enzymes, we characterize beta-thalassemic mutations in 91 homozygous patients and 86 unrelated carriers. Overall, 268 beta-thalassemic genes were obtained. Eleven beta-globin mutations were identified, confirming the wide molecular heterogeneity of beta-thalassemia in Calabria. Information from the present study represents the mainstay for the development of a program of early prenatal diagnosis by direct detection of mutations in Calabria.

GH secretion in thalassemia patients with short stature.

The physiological role of GH secretion on growth retardation remains to be elucidated especially in patients with beta-thalassemia. In the present study, we investigated IGF-1 circulating levels as well as GH release following GHRH alone or combined with some inhibitors of somatostatin: pyridostigmine and arginine. In thalassemic patients lower IGF-1 circulating levels appear to be negatively correlated with both aspartate aminotransferase and alanine aminotransferase as well as with ferritin circulating levels indicating a probable role of hepatic hemosiderosis in IGF-1 production. The authors however suggest that reduced IGF-1 secretion is not the main cause of growth retardation since this would have elicited an enhanced response of GHRH in the presence of a normal hypothalamic pituitary axis. In contrast, they noticed that GH response to GHRH when expressed as area under the curve was lower in thalassemic patients compared to controls. The combination of GHRH with either pyridostigmine or arginine induced a GH secretion in thalassemics which was comparable to that of controls. The results of this study lead to conclude that the alteration of GH secretion is due, in such patients, to an increased somatostatin activity.

Follow-up study of thyroid function in polytransfused thalassemic patients.

The aim of our investigation was to evaluate thyroid function by a follow-up study in 45 polytransfused thalassemic patients, since endocrine abnormalities are frequent consequences of iron overload in thalassemia major. Significant changes of thyroid function have been revealed in the time elapsing the observation, despite unchanged haematological parameters; at the end of the present study five patients were affected by overt hypothyroidism and 15 patients by subclinical hypothyroidism. Ultrasound thyroid volume in 13 randomly selected patients was greatly reduced, while thyroid Magnetic Resonance Imaging (MRI) was not able to detect tissue alterations. Inversely, liver MRI was markedly reduced in 14 patients and negatively related to ferritine levels (P< 0.01). We conclude that polytransfused thalassemics are frequently affected by thyroid disfunction; haepatic haemosiderosis due to iron overload seems influence hormonal peripheral metabolism, although the patients display a moderate compliance with iron chelation therapy. Therefore, periodic thyroid investigation should be carried out in thalassemic subjects in order to detect patients who need hormone replacement therapy.

In vivo metabolic studies of glucose, ATP and 2,3-DPG in beta-thalassaemia intermedia, heterozygous beta-thalassaemic and normal erythrocytes: 13C and 31P MRS studies.

13C and 31P magnetic resonance spectroscopy was used to characterize the in vivo kinetics of glucose metabolism and intracellular ATP and 2,3-DPG concentrations in erythrocytes obtained from beta-thalassaemia intermedia, heterozygous beta-thalassaemic and normal individuals and maintained in suspension. Except for an upfield chemical shift in the 2P and 3P resonance of 2,3-DPG in the thalassaemia intermedia erythrocytes, the 31P spectra were comparable between all three blood types, showing similar concentrations of ATP (from 4.5 to 5.2 mumol/g Hb) and 2,3-DPG (from 17.2 to 19.7 mumol/g Hb). However, the profile of glucose metabolism was quite different in beta-thalassaemia intermedia erythrocytes, whereas glucose was consumed at a rate of 0.089 +/- 0.035 fmol/cell/h, significantly higher than that of normal (0.032 +/- 0.018 fmol/cell/h; P = 0.01) and heterozygous (0.025 +/- 0.004 fmol/cell/h; P = 0.01) erythrocytes. This near 3-fold faster rate of glucose metabolism in the thalassaemia intermedia erythrocytes could not be accounted for by any increase in glucose flux via the Embden-Meyerhof pathway, since no significant difference in 3-13C-lactate synthesis was observed among the three blood types (in units of fmol/cell/h, normal, 0.021 +/- 0.013; heterozygous, 0.021 +/- 0.006; beta-thalassaemia intermedia 0.045 +/- 0.025). These results reflect an accelerated rate of glucose metabolism in thalassaemia intermedia erythrocytes because the contribution of reticulocytes to this altered pattern of metabolism could be excluded. As the only other route of glucose metabolism in erythrocytes is the pentose phosphate pathway (PPP), these results indicate that the PPP is more active in beta-thalassaemia intermedia erythrocytes, perhaps as a consequence of their elevated intracellular oxidative state.

G6PD haplotypes spanning Xq28 from F8C to red/green color vision.

The most telomeric region of the human X chromosome within band Xq28 consists of a gene-rich region of about 3 Mb which contains the genes for coagulation factor VIIIc, glucose-6-phosphate dehydrogenase (G6PD), and red/green color vision. We have studied five polymorphic sites from this region, in a sample of normal people from the Cosenza province of Southern Italy. These sites, which span a distance of some 350 kb, are in strong linkage disequilibrium. Of the 32 possible haplotypes only 10 were found, and 4 of these account for 80% of all X chromosomes analyzed. In addition, we found that all G6PD-deficient people with the G6PD Mediterranean mutation belong to only two haplotypes. One of these (Med 1) is found only within a small subregion of the area investigated, west of the Appennine mountain range. Most remarkably, all Med 1 G6PD-deficient individuals also had red/green color blindness. The more frequent haplotype (Med 2) is the same in Calabria and in Sardinia, where it accounts for about 90% of the G6PD Mediterranean mutations, despite the fact that gene flow between the populations of Sardinia and Southern Italy must have been limited. These data do not enable us to determine whether the two types of G6PD Mediterranean have arisen through two separate identical mutational events or through a single mutational event followed by recombination. However, the data indicate relatively little recombination over an extended region of the X chromosome and they suggest that the G6PD Mediterranean mutation is recent by comparison to the other polymorphisms investigated.

RFLPs of the APOB gene: comparative study between Greeks and southern Italian peoples.

Restriction fragment length polymorphisms (RFLPs) at codons 2488 (XbaI), 3611 (MspI), and 4154 (EcoRI) of the apolipoprotein B gene were investigated in sample groups from Athens (Greece) and Calabria (southern Italy) to verify whether the distribution of the APOB gene variants in Calabria, where Greek colonization occurred in the eighth century B.C., reflects that of the present Greek population. A sample from Apulia, a southern Italian region having a history different from that of Calabria, was also analyzed. Three specific DNA regions, each containing the polymorphic site, were amplified by polymerase chain reaction on 243 samples, and the restriction data for the three groups were compared. The allelic frequencies of the samples from Apulia and Greece showed variability patterns that agree with those found in Caucasians, whereas the Calabrian sample shows remarkable peculiarities, mainly for the EcoRI RFLP. Linkage disequilibrium analyses of pairs of markers showed strong D linkage values between X-M markers, whereas the D linkage values between M-R markers were too small to be reliably estimated. Last, for both Apulians and Greeks, X-R markers showed linkage disequilibrium, whereas for Calabrians they did not. Estimates of XMR haplotypic frequencies were computed; they were found to be appreciably different between Calabrian and Greek samples, whereas the frequencies in the Apulian sample were approximately midway between those in Calabrians and Greeks.

Genetic heterogeneity of glucose-6-phosphate dehydrogenase deficiency revealed by single-strand conformation and sequence analysis.

We have carried out a systematic study of the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on a sample of 53 male subjects from Calabria, in southern Italy. Our sequential approach consisted of the following steps: (1) Partial biochemical characterization was used to pinpoint candidate known variants. The identity of these was then verified by restriction-enzyme or allele-specific oligonucleotide hybridization analysis of the appropriate PCR-amplified fragment. (2) On samples for which there was no obvious candidate mutation, we proceeded to amplify the entire coding region in eight fragments, followed by single-strand conformation polymorphism (SSCP) analysis of each fragment. (3) The next step was M13 phage cloning and sequencing of those individual fragments that were found to be abnormal by SSCP. Through this approach we have identified the molecular lesion in 51 of the 53 samples. In these we found a total of nine different G6PD-deficient variants, five of which (G6PD Mediterranean, G6PD A-, G6PD Coimbra, G6PD Seattle, and G6PD Montalbano) were already known, whereas four are new (G6PD Cassano, G6PD Cosenza, G6PD Sibari, and G6PD Maewo). G6PD Mediterranean is the commonest variant, followed by G6PD Seattle. At least seven of the variants are present, at polymorphic frequencies, in the Calabria region, and some have a nonrandom distribution within the region. This study shows that the genetic heterogeneity of G6PD deficiency in Calabria, when analyzed at the DNA level, is even greater than had been anticipated from biochemical characterization. The sequential approach that we have followed is fast and efficient and could be applied to other populations.